Disposable bioreactor system

ABSTRACT

A cylindrical or annular polymeric bioreactor is disclosed which provides enhanced mixing and aeration of the growth medium while simultaneously offering reduced mechanical shear force.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/008,447, now U.S. Pat. No. 9,868,095, filed on 10 Jan. 2008, entitled“DISPOSABLE BIOREACTOR SYSTEM,” which application claims benefit under35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 60/927,274filed May 2, 2007, entitled “DISPOSABLE BIOREACTOR SYSTEM,” all of whichapplications are herein incorporated by reference in their entiretiesand for all purposes.

FIELD OF THE INVENTION

This invention relates to an improved design for a disposable bioreactorvessel suitable for cell culture and fermentation.

BACKGROUND OF THE INVENTION

The increasing popularity of disposable or single use bioreactors forupstream processing has been recently noted in several studies, and canbe understood by considering a typical biotech manufacturing facility.The infrastructure required to implement a facility using traditionalglass/steel bioreactors is substantial, and the time and expenserequired to construct it can be immense. The requirement that both thebioreactor itself, and also the ingress and egress tubing, utilize inertmaterials such as 316L electro-polished stainless steel requires a largeinitial investment. Additionally, the size and form factor of disposablebioreactor vessels generally lend themselves to easier storage andre-configurability when compared with traditional, rigid glass/steelsolutions. Other advantages are the cost and time savings overtraditional designs, the reduction in preparation and sterilizationtime, the reduced need for purified water for cleaning the vessel aftera run, and the significantly reduced post run maintenance time.Additionally, single use bioreactors and the associated plastic tubinglend themselves to being re-configured and validated quickly andefficiently as manufacturing or process requirements change. Although anumber of different styles of single use bioreactors have been conceivedand introduced into the marketplace, two types currently predominate.

The first type of single-use bioreactor is generally referred to as the“pillow” or “rocker” bag style, and is described, for example, in U.S.Pat. No. 6,190,913. This style of bag has been constructed from avariety of different polymeric materials, but low density polyethyleneand ethylene/vinyl-acetate copolymers are currently the most popularmaterials for at least the innermost layer which contacts the aqueousgrowth medium. This type of disposable bioreactor utilizes a wave motioninduced by movement about a single axis to both mix and sparge (aerate)the contents of the bioreactor.

Another style of bioreactor bag is often referred to as a “liner style”and substantially mimics the function and form of a traditionalglass/steel bioreactor. A disposable polymeric bag is used as a linerinside a generally cylindrical glass/steel tank and generally uses animpeller to mix the contents of the bioreactor vessel. This type ofsystem has been commercialized by several manufacturers (see e.g.,Published US Patent Applications 2005/0272146 and 2005/0239199). Thedisposable liner type of bioreactor bag has proven popular for processdevelopment or pilot runs using growth medium volumes of 25 liters ormore.

Both styles of disposable bioreactors have undergone some scrutiny inorder to judge their efficacy in comparison to traditional glass/steelbioreactors. However, statistically rigorous analyses are apparently notavailable to date. Irrespective of the style of the disposablebioreactor, the inner surface of the polymeric bioreactor bag needs tobe both biologically inert and also not prone to leaching nutrients fromthe growth medium or from the polymer into the growth medium (seeKadarusman et. al, Growing Cholesterol-Dependent NSO Myeloma Cell Linein the Wave Bioreactor System: Overcoming Cholesterol-PolymerInteraction by using Pretreated Polymer or Inert Fluorinated EthylenePropylene, Biotechology Progress, 2005, 21, p. 1341). Additionally, theliner needs to be chemically stable under the optical illumination oftenused to facilitate cell growth.

Whether a bioreactor is of traditional design or a modern disposableformat, the basic function of a bioreactor is to provide a controlledenvironment in order to achieve optimal growth/product formation in thecell or microbe that is present in the aqueous bioreactor growth medium.The traditional glass and steel bioreactors used in batch processes wereproven to be effective in the course of antibiotic production in the1950's, and still remain the dominant paradigm in the fermentationindustry. Criteria for optimizing bioreactor design have been enumeratedbefore (see A. Margaritas and J. B. Wallace, Novel Bioreactor Systemsand Their Applications, Nature Bio/Technology, May 1984, p. 447.)According to Margaritas et al, some of the basic criteria required tocharacterize and understand bioreactor performance are:

1. Mass and heat transfer and hydrodynamic characteristics of thebioreactor,

2. Potential for bioreactor scale-up, and

3. Capital and operating costs of the bioreactor

Given the extensive existing knowledge regarding the design andperformance of traditional glass/steel bioreactors, it was considereddesirable to mimic their design as much as possible in theimplementation of a disposable bioreactor system. Examination of currentproducts reveals that this approach has been utilized, but with onlymixed results. An example of this prior art style of product, as shownin FIG. 1, employs a liner made of a bio-compatible, flexible polymericmaterial set inside a vessel which provides structural support. The endresult is a disposable liner which assumes a physical form very similarto that of a traditional (generally cylindrical) bioreactor. The use ofan impeller and sparger completes the analogy to a traditionalglass/steel vessel. Therefore, the performance of a liner typedisposable bioreactor can be measured against the list of criteria 1through 3 above, and one can get a reasonable idea of the performancethrough comparison to the performance of similar size and shapetraditional glass/steel systems. Given the analogy to known bioreactorshapes and sizes, impeller design and placement, the mixing andoxygenation rates can be approximated. Additionally, traditional scalingarguments can be formulated and implemented, and known techniques ofcomputational fluid mechanics can be applied.

The “pillow” bag style of disposable bioreactor also utilizes bags madeof biocompatible films. An example of this design is shown in FIG. 2(see V. Singh, Disposable Bioreactor for cell culture using wave-inducedagitation, Cytotechnology 30, 1999, p. 149.). Most proponents of the“pillow” bag stipulate that traditional bioreactors and single-usebioreactors that emulate the traditional glass/steel bioreactor using adisposable liner limit the yield obtainable in mammalian cell growthruns due to impeller induced sheer stress of the cells during mixing andsparging. The “pillow” bag style of disposable bioreactor bag has noimpeller, and utilizes a rocking motion to mix and sparge themicro-organisms contained within the bag. It is claimed that thisrocking motion is gentle on the cells and does not cause sheer stresswhich can easily damage mammalian cells (G. Kretzmer and K. Schugerl,Journal of Applied Microbiology and Biotechnology, Vol. 34, No. 5,613-616 (1991)). The boundary conditions set forth by the bag, combinedwith the rocking motion are alleged to adequately mix and aerate theaqueous culture medium when mammalian cells are used. However, it is notentirely clear that this is the case or that this style of bioreactor isefficacious for non-mammalian cell processes, such as bacterial ormicrobial fermentations that require more oxygen and/or can have highlyviscous contents (reaction medium).

As shown in FIG. 2, the most common commercial versions of the pillowbag style single-use bioreactor utilizes one or more ports on the top ofthe bag to bring in the air or oxygen, and then utilizes another(output) port in relatively close proximity to the input port to ventthe gas(es) produced by the bioprocess. It is likely that in many casesthe incoming sparging gas is vented too quickly so that sub-optimaloxygenation occurs. For this type of bag, no evidence of anunderstanding of the performance criteria 1 through 3, as set forthabove, is found in the literature. Published data generally indicatesthat the total cell density achieved in this style of bioreactor isgenerally less than (and at best, equal to) that achieved in smallshaker flasks.

Another style of disposable bioreactor as made by Corning is shown inFIG. 1. This product is a disposable or single-use spinner flask. Thisflask is made of a rigid biocompatible plastic and uses a magneticallydriven internal impeller to mix the contents. However, this design isonly suitable for relatively small volume growth processes.

It is an object of the present invention to provide a design for asingle-use bioreactor vessel which ensures adequate mixing of theingredients in the growth medium, and sufficient aeration for cells ormicrobes which require significant oxygen, but at the same time does notcause destructive shear stress and is scalable to comparatively largevolume runs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 shows the prior art type of disposable bioreactor system (e.g.,Hyclone/Thermo-Fisher) that utilizes a disposable polymeric liner andprovides structural support for the liner.

FIG. 2 shows a prior art “pillow” disposable bioreactor bag of flexiblepolymeric material.

FIG. 3a shows a three-dimensional view of a bioreactor in accordancewith the present invention having vertical side walls and which istoroidal in shape.

FIG. 3b shows two-dimensional views of a toroidal bioreactor inaccordance with the present invention as depicted in FIG. 3a and showsalternative embodiments of the vessel having either a circular (12) orrectangular (11) cross-section.

FIG. 3c shows how the bioreactor of the present invention achieves itsenhanced mixing via orbital motion (in this case moving counterclockwise indicated as 16) which creates a travelling wave of the growthmedium present in the bioreactor vessel (17).

FIG. 4 shows a view of the walls of the preferred embodiment of thebioreactor vessel of FIGS. 3a and 3b where sparging is accomplishedthrough the walls of the vessel. The walls are shown as corrugated inorder to enhance mixing.

FIG. 5 shows a view of an alternative configuration for a bioreactorvessel in accordance with the present invention where the bioreactor istoroidal but oval in cross-section rather than circular. Again, thevessel can have a cross-section that is either substantially rectangular(5.2) or circular (5.3).

FIG. 6 shows an embodiment of the present invention which provides formulti-directional motion (i.e., both orbital and rocking) of thebioreactor vessel of the present invention shown in FIG. 3a , with the Xand Y axes shown.

DESCRIPTION OF THE INVENTION

The most desirable single-use bioreactor solution would be one thateffectively meets criteria 1 through 3, as set forth above, while at thesame time requiring the fewest moving parts. Minimizing the number ofmoving parts will also tend to yield the least expensive and mostreliable overall solution. A novel design for a polymeric bioreactorvessel in accordance with the present invention which uses a minimalnumber of parts is shown in FIGS. 3 through 6.

In FIG. 3a , a cylindrical bioreactor vessel 1 having a substantiallyrectangular cross-section is shown having a hollow core 6, so that thebioreactor is a toroid. Although the design shown in FIG. 3a is shown ashaving substantially vertical walls, this is not an essential aspect ofthe invention and a toroidal (donut) shaped vessel not having verticalsurfaces is also suitable, as shown in FIG. 3b in cross section as 12.Alternatively, only one wall (either inner or outer) may be vertical andthe other curved.

The vessel can suitably be of a rigid biocompatible polymer.Alternatively, the vessel can be fabricated in whole or in part of aflexible (non-rigid) polymer since the hydrostatic pressure of theliquid reaction (growth) medium present within the vessel together withthe pressure of the sparging oxygen or oxygen containing gas will enableeven a bag made of non-rigid polymer to substantially retain its annularshape. Suitable rigid polymers include, but are not limited to; USPClass VI approved polycarbonate and polystyrene. Suitable flexiblepolymers include, but are not limited to, low density polyethylene andethylene/vinyl acetate copolymer.

In FIG. 3a , element 2 is a platform equipped with an electric motor orother drive means (not shown) which moves in an orbital motion asindicated by arrow 3 and as further shown in FIG. 3c . This orbitalmotion, combined with the toroidal shape of the bioreactor, sets up atraveling wave in the bioreactor growth medium. This traveling waveprovides both effective mixing and oxygenation of the contents. Itshould be noted that in the case of a torioidal vessel whether havingvertical or curved walls, the liquid motion will follow a pure travelingwave. In both cases, the exact liquid motion can be modeled usingcomputational fluid dynamics.

Additionally, single-use bioreactor vessel 1 can suitably be equippedwith one or a plurality of input ports (one shown as 4 in FIG. 3a ) forprocess feedstock inputs (e.g.: pH buffers, glucose etc.), and/or alsofor pre-inserted and pre-calibrated sensors (e.g., to measure dissolvedoxygen, pH, dissolved CO.sub.2 and the like). These sensors can beeither traditional electrochemical sensors and/or disposable andpre-calibrated optical sensors. It is generally more efficacious to havethe sensors pre-calibrated in order to avoid breaking the sterilebarrier on the bioreactor. Optical probes are available that can be bothpre-calibrated and can be gamma irradiated while mounted in thebioreactor. The ability to use gamma radiation allows the pre-calibratedsensors to be inserted in the bioreactor and the entire assemblysubsequently sterilized. Port 5 denotes the exit port for removal ofgaseous reaction by-products. In FIG. 3a 7 denotes a light source suchas a bank of visible or UV light emitting LEDs that can be utilized topromote the growth of plant or algae culture or transiently activatecertain genes for multi-drug production from a single cell platform Thelighting is shown on the top, but this is not a necessity; theillumination can additionally or alternatively come from the sidesand/or even the bottom of the vessel provided only that the growth mediareceives enough illumination to enhance photosynthesis or geneactivation depending on the purpose of the growth run and the cell beinggrown. Ideally the LEDs will have a spectrum that is matched to theabsorption spectrum of the species being grown. LEDs are now availablefrom the ultra-violet through the near infrared so matching to theabsorption spectrum of the species under study can be easily done.Additionally, the surface of the bioreactor on which the LED's aremounted needs to be sufficiently transmitting in this region to allowthe light to pass through to the interior of the bioreactor.

The bioreactor vessel walls (outer and optional inner surfaces) define astructure that will have a resonant frequency which is determined by theparticular configuration (size and shape) of the bioreactor. Theresonant frequency of the bioreactor can be readily calculated knowingthat the traveling wave must reproduce itself in phase every round trip(Hydrodynamics, Horace Lamb and Russ Caflisch, First CambridgeUniversity Press, 1997). Once this frequency has been determined thebioreactor can be rotated with this circular frequency to thereby set-upresonant traveling wave motion of the fluid (growth medium) inside. Thewave amplitude will be chosen depending on the level of mixing andagitation needed.

In FIG. 3b the top view, 10, of the bioreactor is shown along with thesquare or alternatively circular cross sections 11 and 12 respectively.

It is possible to further enhance the mixing and sparging efficiency ofthe bioreactor vessel of the present invention shown in FIG. 4 byproviding a textured surface on the inner floor (bottom) and/or wallsurfaces of the vessel (i.e., a surface which is in contact with thegrowth medium). This texturing can be achieved using baffles, ridges,bumps and/or other upstanding protuberances as shown in FIG. 4, whichare preferably pre-molded on all or a part of the inner surface of thevessel. The sparging of the reaction medium is preferably accomplishedin this design by bringing the oxygen or air into the bioreactor througha multitude of orifices in the floor and/or walls of the vessel. Theseorifices can be in the protuberances in the texturing or separate fromthem. In FIG. 4, the flow direction of the fluid in the annular vesselis indicated as 4.1, (which corresponds to arrow 3 in FIG. 4), thetextured surface on the inner wall and/or floor surface of the vessel(the textured surface being shown here for simplicity to have anapproximately sinusoidal shape pattern) is indicated as 4.2, and thesparge orifices are indicated as 4.3.

The optimal patterning (e.g., size, shape and frequency) will be afunction of the size of the reactor, the velocity, viscosity, and natureof cell platform and its associated optimized growth medium. Theparticular patterning which provides optimal agitation can be determinedthrough finite element analysis studies (www.fluent.com) or throughempirical experiment. These studies generally include mixing studies asa function of time or number of agitation cycles. Additionalcomputational studies that employ Henry's law (p. 384, GeneralChemistry, 2^(nd) Edition, Donald A. McQuarrie and Peter A. Rock, W. H.Freeman and Company, New York, 1987) to model oxygen transfer orcalculate the oxygen transfer rate are possible. These studies requirethe finite element analysis code to take into account the surface areaof the bubbles created during sparging. For example, a higher number ofbubbles having decreased size will increase the surface area availablefor oxygen transfer.

In FIG. 4, the sparging gas (normally air or oxygen) is suitably broughtto the bioreactor using tubing through the sparge orifices 4.3 whichpreferably will have a fluid diode or hydrophobic filter at or near itsend that allows the sparging gas to flow into the bioreactor, butprevents the liquid (reaction medium) from draining out of thebioreactor vessel through said tubing. The aforementioned texturing ofthe floor and/or walls therefore does not preclude bringing the spargegas into the vessel through the floor, top, and/or walls of the vessel.This type of sparging, combined with the traveling wave motion inducedby the orbital movement of the vessel, ensures that the cells ormicrobes in the bioreactor have access to sufficient feed and oxygen toachieve optimal total cell density and/or maximize product yield.

It should also be noted that the motion in direction FIG. 3a is onlyshown as orbital, but can also involve rocking in either the X or Y axis(or both) and thereby be a two-dimensional or three-dimensional motion,which can be readily tailored to work in conjunction with the bioreactordimensions. Such multi-directional motion is shown in FIG. 6 where 6.1and 6.2 are the plane of the platform when rocked about axis 6.3. Theplatform holding the bioreactor can also be orbited in one directionaccording to 6.4, or agitated in both directions as per 6.5. This motioncan also be a reciprocating motion where the direction of rotation ofthe motor which causes the vessel to orbit is periodically reversed.This periodic reversal in the direction of the vessel's orbit can beused to cause more vigorous agitation if/when required during the growthprocess.

The invention claimed is:
 1. An apparatus comprising: (a) a hollowtoroidal one-piece polymeric bioreactor vessel comprising: at least oneport configured to receive at least one sensor to monitor a bioprocesscarried out in said hollow toroidal one-piece polymeric bioreactorvessel; textured surface arranged on an inner surface of the hollowtoroidal one-piece polymeric bioreactor vessel, wherein a sinusoidalshape pattern of the textured surface comprises a plurality ofupstanding protuberances; and a multitude of tubes coupled to the innersurface and extending through the plurality of upstanding protuberancesof the textured surface, wherein the multitude of tubes are configuredfor sparging gas into said hollow toroidal one-piece polymericbioreactor vessel; and (b) a drive including a motor driven platform,wherein when in operation the hollow toroidal one-piece polymericbioreactor vessel rests on the motor driven platform and the platformmoves orbitally such that there is build-up of a resonant frequencytraveling wave of fluid orbiting in one direction in an interior of thehollow toroidal one-piece polymeric bioreactor vessel when a particularamplitude and orbital speed is imparted to said hollow toroidalone-piece polymeric bioreactor vessel, wherein the resonant frequencydepends on size and shape of the hollow toroidal one-piece polymericbioreactor vessel, wherein the hollow toroidal one-piece polymericbioreactor vessel was sterilized by gamma radiation, and wherein when inoperation the textured surface including the multitude of tubes sparginggas into said hollow toroidal one-piece polymeric bioreactor vessel andthe traveling wave of fluid induced by the orbital movement of thehollow toroidal one-piece polymeric bioreactor vessel are configured toenhance aeration and mixing efficiency on the inner surface of thehollow toroidal one-piece polymeric bioreactor vessel.
 2. The apparatusin accordance with claim 1, wherein the textured surface is located on abottom of the hollow toroidal one-piece polymeric bioreactor vessel. 3.The apparatus in accordance with claim 2, wherein said hollow toroidalone-piece polymeric bioreactor vessel includes at least one exit portfor removal of gaseous by-products.
 4. The apparatus in accordance withclaim 1, wherein the sinusoidal shape pattern of the textured surface isformed of pre-molded baffles.
 5. The apparatus in accordance with claim1, wherein the hollow toroidal one-piece polymeric bioreactor vessel isfabricated from a rigid plastic.
 6. The apparatus in accordance withclaim 1, wherein the hollow toroidal one-piece polymeric bioreactorvessel is fabricated from a flexible plastic.
 7. The apparatus inaccordance with claim 1, further comprising means for illuminatingcontents of said hollow toroidal one-piece polymeric bioreactor vessel.8. The apparatus in accordance with claim 7, wherein said means forilluminating the contents of said hollow toroidal one-piece polymericbioreactor vessel comprises at least one LED.
 9. The apparatus inaccordance with claim 1, wherein the drive is further configured forsimultaneously both orbiting and rocking said hollow toroidal one-piecepolymeric bioreactor vessel.
 10. The apparatus in accordance with claim1, further comprising a plurality of input ports for inserting a sensorsor process feedstock inputs into the hollow toroidal one-piece polymericbioreactor vessel.
 11. The apparatus in accordance with claim 1, whereinthe at least one sensor is a pre-inserted and pre-calibrated sensor. 12.The apparatus in accordance with claim 1, wherein the drive isconfigured to move the hollow toroidal one-piece polymeric bioreactorvessel in a reciprocating motion to cause the fluid to orbit in areversed direction.
 13. An apparatus comprising: (a) a hollow toroidalone-piece polymeric bioreactor vessel comprising: at least one portconfigured to receive at least one sensor to monitor a bioprocesscarried out in said hollow toroidal one-piece polymeric bioreactorvessel; a textured surface arranged on an inner bottom surface of thehollow toroidal one-piece polymeric bioreactor vessel, wherein asinusoidal shape pattern of the textured surface comprises a pluralityof upstanding protuberances; and a multitude of tubes extending throughthe inner bottom surface for sparging gas into said hollow toroidalone-piece polymeric bioreactor vessel; and (b) a drive including a motordriven platform, wherein when in operation the hollow toroidal one-piecepolymeric bioreactor vessel rests on the motor driven platform and theplatform moves orbitally such that there is build-up of a resonantfrequency traveling wave of fluid orbiting in one direction in aninterior of the hollow toroidal one-piece polymeric bioreactor vesselwhen a particular amplitude and orbital speed is imparted to said hollowtoroidal one-piece polymeric bioreactor vessel, wherein the resonantfrequency depends on size and shape of the hollow toroidal one-piecepolymeric bioreactor vessel, wherein the hollow toroidal one-piecepolymeric bioreactor vessel was sterilized by gamma radiation, whereinwhen in operation the textured surface, the multitude of tubes sparginggas into said hollow toroidal one-piece polymeric bioreactor vessel, andthe traveling wave of fluid induced by the orbital movement of thehollow toroidal one-piece polymeric bioreactor vessel are configured toenhance aeration and mixing efficiency on the inner bottom surface ofthe hollow toroidal one-piece polymeric bioreactor vessel.
 14. Theapparatus in accordance with claim 13, wherein the at least one sensoris a pre-inserted and pre-calibrated sensor.